We have investigated the function of the prominently represented HLA molecules on T lymphocytes by studying the synthesis and turnover of HLA-A,B,C molecules in human peripheral lymphocytes. Newly synthesized HLA-A,B,C and beta2-microglobulin molecules were identified among total lymphocyte proteins by two-dimensional gel electrophoresis and specific immunoselection. In resting T cells, the polymorphic set of HLA-A,B,C proteins was among the most prominently synthesized and rapidly turned over of total cell proteins and was the most rapidly turned over of all plasma membrane proteins. Following growth stimulation, the rate of HLA synthesis was increased to a lesser degree than total protein synthesis, but turnover was reduced markedly. The net result was accumulation of HLA molecules in the plasma membrane. We suggested that continuous degradation and replacement reflects the biochemical nature of the participation of HLA molecules in immunological surveillance by quiescent lymphocytes, while cessation of turnover and accumulation of surface HLA molecules is a biochemical adjustment related to T cell activation. With improved technique, we have been able to characterize the specific class I HLA gene products of particular individuals. Using this approach, we studied 5 subjects whose HLA-A,B antigens were indistinguishable by serological tests, but some whom showed mutual immunological responses indicating functional differences in certain HLA proteins. In every case where a functional difference was detected, we observed specific structural differences in HLA proteins, evidenced by altered molecular charge. Where functional differences were absent, no molecular charge differences were detected. Based on our findings, we proposed that amino acid substitutions which generate altered molecular charge may be an important factor in the generation of HLA polymorphism detectable by cytotoxic T lymphocytes. Moreover, these studies suggest that charged amino acids may play a significant role in the biochemical mechanism by which T cells distinguish self from non-self histocomopatibility antigens.